Semicarbazide-sensitive amine oxidase (SSAO), also known as plasma amine oxidase and benzylamine oxidase, is identical in structure to vascular adhesion protein-1 (VAP-1). In the following discussion, SSAO/VAP-1 is used to describe this protein. The role of this protein in inflammatory diseases has been reviewed (see, for example, Smith D. J. and Vaino P. J., Targeting Vascular Adhesion Protein-1 to Treat Autoimmune and Inflammatory Diseases. Ann. N.Y. Acad. Sci. 2007, Vol 1110, pages 382-388; and McDonald I. A. et al., Semicarbazide Sensitive Amine Oxidase and Vascular Adhesion Protein-1: One Protein Being Validated as a Therapeutic Target for Inflammatory Diseases. Annual Reports in Medicinal Chemistry, 2008, Vol 43).
In most organisms, including humans, two families of mammalian amine oxidases metabolize various mono-, di-, and polyamines produced endogenously or absorbed from exogenous sources. These include the monoamine oxidases (MAO-A and MAO-B) which are present in the mitochondria of most cell types and use covalently bound flavin adenine dinucleotide (FAD) as the cofactor. Polyamine oxidase is another FAD-dependent amine oxidase which oxidatively deaminates spermine and spermidine. SSAO/VAP-1 belongs to the second family which is dependent on copper and uses other co-factors apart from FAD, such as an oxidized tyrosine residue (abbreviated as TPQ). MAO and SSAO/VAP-1 oxidatively deaminate some common substrates which includes the monoamines such dopamine, tyramine and benzylamine.
Some of these enzymes were originally defined by the ability of certain compounds to inhibit the enzymatic activity thereof For example MAO-A is selectively inhibited by clorgyline, MAO-B by L-deprenyl, while neither clorgyline nor L-deprenyl can inhibit the amine oxidase activity of SSAO/VAP-1. SSAO/VAP-1 can be inhibited by semicarbazide, hence the name semicarbazide sensitive amine oxidase.
SSAO/VAP-1 is an ectoenzyme containing a very short cytoplasmic tail, a single transmembrane domain, and a large, highly glycosylated extracellular domain which contains the active center for the amine oxidase activity. SSAO/VAP-1 is also present in a soluble form circulating in the plasma of some animals. It has been shown that this form is a cleaved product of membrane-bound SSAO/VAP-1. It is currently unclear if there are distinct functions for the membrane and soluble form of SSAO/VAP-1.
SSAO/VAP-1 appears to have two physiological functions: the first is the amine oxidase activity mentioned above and the second is cell adhesion activity. Both activities are associated with inflammatory processes. SSAO/VAP-1 was shown to play an important role in extravasation of inflammatory cells from the circulation to sites of inflammation (Salmi M. and Jalkanen S., VAP-1: an adhesin and an enzyme. Trends Immunol 2001, 22, 211-216). VAP-1 antibodies have been demonstrated to attenuate inflammatory processes by blocking the adhesion site of the SSAO/VAP-1 protein, and, together with a substantial body of evidence of in vitro and in vivo knockouts, it is now clear that SSAO/VAP-1 is an important cellular mediator of inflammation. Transgenic mice lacking SSAO/VAP-1 show reduced adhesion of leukocytes to endothelial cells, reduced lymphocyte homing to the lymph nodes and a concomitant attenuated inflammatory response in a peritonitis model. These animals were otherwise healthy, grew normally, were fertile, and examination of various organs and tissues showed the normal phenotype. Furthermore, inhibitors of the amine oxidase activity of SSAO/VAP-1 have been found to interfere with leukocyte rolling, adhesion and extravasation and, similar to SSAO/VAP-1 antibodies, exhibit anti-inflammatory properties.
Inflammation is the first response of the immune system to infection or irritation. The migration of leukocytes from the circulation into tissues is essential for this process. Inappropriate inflammatory responses can result in local inflammation of otherwise healthy tissue which can lead to disorders such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis and respiratory diseases. Leukocytes first adhere to the endothelium via binding to adhesion molecules before they can start the process of passing through the walls of the blood vessels. Membrane bound SSAO/VAP-1 is abundantly expressed in vascular endothelial cells such as high venule endothelial cells (HVE) of lymphatic organs and is also expressed in hepatic sinusoidal endothelial cells (HSEC), smooth muscle cells and adipocytes. The expression of SSAO/VAP-1 on the cell surface of endothelial cells is tightly regulated and is increased during inflammation. In the presence of an SSAO/VAP-1 substrate (benzylamine), NFκB was activated in HSECs together with up regulation of other adhesion molecules, E-selectin and chemokine CXCL8 (IL-8) in vitro. A recent study confirms this result by showing by mutagenesis that the transcription and translation of E-selectin and P-selectin is induced by the enzyme activity of SSAO/VAP-1. These results suggest an important role of the amine oxidase activity of SSAO/VAP-1 in the inflammatory response. It has been reported that the oxidase activity of SSAO/VAP-1 induces endothelial E- and P-selectins and leukocyte binding (Jalkanen S, et al., The oxidase activity of vascular adhesion protein-1 (VAP-1) induces endothelial E- and P-selectins and leukocyte binding. Blood 2007, 110, 1864-1870).
Excessive and chronic inflammatory responses have been associated with the symptoms of many chronic diseases, such as rheumatoid arthritis, multiple sclerosis, asthma, bronchitis and chronic obstructive pulmonary disease (COPD). Patients suffering from either atopic eczema or psoriasis (both chronic inflammatory skin disorders) have higher levels of SSAO/VAP-1 positive cells in their skin compared to skin from healthy controls.
SSAO/VAP-1 is also highly expressed in adipocytes where it plays a role in glucose transport independent of the presence of insulin. It has been observed that levels of plasma SSAO/VAP-1 are increased in patients suffering from diabetes. Elevated levels of plasma SSAO/VAP-1 have been found in patients suffering from other illnesses, such as congestive heart failure and liver cirrhosis. It is believed that SSAO/VAP-1 is associated with most, if not all, inflammatory diseases whether the inflammation is in response to an immune response or subsequent to other events such as occlusion and reperfusion of blood vessels.
It has been reported that inhibition of vascular adhesion protein-1 (VAP-1) suppresses endotoxin-induced uveitis (EIU). In the retina, SSAO/VAP-1 is exclusively expressed in the vasculature, and its expression level was found to be elevated during EIU. SSAO/VAP-1 inhibition in animal models of EIU significantly suppressed leukocyte recruitment to the anterior chamber, vitreous, and retina. The data suggest an important role for SSAO/VAP-1 in the recruitment of leukocytes to the immune-privileged ocular tissues during acute inflammation (see, for example, Noda K. et al., Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis. Faseb J22, 1094-1103 (2008)) and Noda K. et al., A Vascular adhesion protein-1 blockade suppresses choroidal neovascularization. Faseb J (2008)).
In a mouse overexpressing human SSAO/VAP-1, the SSAO inhibitor Mofegiline significantly attentuates the LPS-induced increase in TNF-a. This inhibitor also significantly reduces BAL cell counts. These results demonstrate that SSAO/VAP-1 is involved in LPS-induced pulmonary inflammation (see, for example, P. Yu et al American Journal of Pathology, 168, 718-726 (2006)). Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are serious inflammatory disorders of the lung. These disorders are thought to develop when pulmonary or systemic inflammation leads to systemic release of cytokines and other proinflammatory molecules, which recruit neutrophils to the lungs, which in turn release leukotrienes, oxidants, platelet-activating factor, and proteases (see, for example, the Merck Manuals Online Medical Library on the World Wide Web at merck.com/mmpe/sec06/ch066/ch066a.html).
SSAO/VAP-1 is upregulated in the liver and serum of patients with inflammatory liver disease; inhibition of SSAO/VAP-1 activity decreases migration of normal lymphocytes across hepatic sinusoidal endothelial cells and has significant effects on the migration of peripheral blood lymphocytes. It has been suggested that the restricted expression of SSAO/VAP-1 and increased production of secreted SSAO/VAP-1 in inflammatory liver disease indicates utility of this protein as a therapeutic target for diseases associated with liver inflammation (see, for example, P. Lalor et al., Ann. N.Y. Acad. Sci. 1110: 485-496 (2007)).
During the SSAO/VAP-1 amine oxidase catalytic cycle, the covalently bound cofactor, TPQ, is first reduced, and then re-oxidized by oxygen in the presence of copper with the generation of hydrogen peroxide as a by-product. It has been speculated that excessive amounts of hydrogen peroxide concentrations can be deleterious and may contribute to the pathology of various inflammatory and neurodegenerative processes (Götz M. E., et al., Oxidative stress: free radical production in neural degeneration. Pharmacol Ther 1994; 63: 37-122).
Monoamine oxidase inhibitors have been used as therapeutic agents for many years (see Tipton, K. F. et al., Monoamine Oxidase and Disease, Academic Press, 1984). Monoamine oxidase-B (MAO-B) is an enzyme present on outer mitochondrial membranes. In man MAO-B oxidatively deaminates dopamine, N-methylhistamine and some trace amines. Monoamine oxidase inhibitors have been known for many years and some are clinically prescribed medications for the treatment of Parkinson's disease and more recently for the treatment of CNS disorders such as bipolar depression and attention deficit hyperactivity disorder (ADHD), where dopamine is likely to play an important role in the pathophysiology. The most well known MAO-B inhibitor is Selegiline (L-deprenyl, Eldepryl® or Anipryl® for veterinary use) which is used to treat Parkinson's disease and senile dementia. A transdermal patch formulation of Selegiline (Emsam Patch) has been shown to be effective treatment for major depression (Bodkin, J. A. and Amsterdam, J. D., Transdermal Selegiline in Major Depression: A Double-Blind, Placebo-Controlled, Parallel-Group Study in Outpatients. Am J Psychiatry 2002; 159:1869-1875), and is currently being evaluated in ADHD patients. The drug is also being evaluated to help people stop smoking tobacco and marijuana.
Inflammation is believed to be an important feature of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease and multiple sclerosis, and similarly is a feature of the pathophysiology that occurs after a cerebral occlusion/reperfusion event (Aktas, O. et al., Neuronal damage in brain inflammation. Arch Neurol 2007, 64:185-9). Excessive activity of MAO-B and SSAO/VAP-1 has been independently implicated in these processes (Xu, H-L. et al., Vascular Adhesion Protein-1 plays an important role in postischemic inflammation and neuropathology in diabetic, estrogen-treated ovariectomized female rats subjected to transient forebrain ischemia. Journal Pharmacology and Experimental Therapeutics, 2006, 317: 19-26 and Youdim, M. B., Buccafusco, J. J., Multi-functional drugs for various CNS targets in the treatment of neurodegenerative disorders. Trends Pharmacol Sci, 2005 26:27-35).
Some known MAO inhibitors also inhibit SSAO/VAP-1 (e.g., the MAO-B inhibitor Mofegiline illustrated below). Mofegiline has been reported to inhibit experimental autoimmune encephalomyelitis (US 2006/0025438 A1). This inhibitor is a member of the haloallylamine family of MAO inhibitors; the halogen in Mofegiline is fluorine. Fluorallylamine inhibitors are described in Bey P., U.S. Pat. No. 4,454,158 (Allyl amine MAO inhibitors). There have been reports of a chloroallylamine, MDL72274 (illustrated below), selectively inhibiting rat SSAO/VAP-1 compared to MAO-A and MAO-B.
Other examples structurally related to Mofegiline can be found in LJPC WO 2007/120528 A2.
References to the following examples of SSAO/VAP-1 inhibitors, and to the effects of such inhibitors in various animal models of disease, can be found in the review publication by McDonald I. A. et al., Semicarbazide Sensitive Amine Oxidase and Vascular Adhesion Protein-1: One Protein Being Validated as a Therapeutic Target for Inflammatory Diseases. Annual Reports in Medicinal Chemistry, 2008, Vol 43.
A family of modestly potent propargylamine compounds was reported to inhibit SSAO/VAP-1 from a number of diverse species (O'Connell, K. M. et al., Differential inhibition of six copper amine oxidases by a family of 4-(aryloxy)-2-butynamines: Evidence for a new mode of inactivation. Biochemistry 2004, 43, 10965-10978 and WO2007/005737) with activity in the micromolar range. Other SSAO/VAP-1 inhibitors have appeared in the patent literature in recent times. Many of these compounds, like semicarbazide, rely upon a hydrazine functional group to form a covalent imine bond with the TPQ cofactor. Two examples are presented below and can be found in the patent publications from La Jolla Pharmaceutical Company (LJPC, WO 2006/094201) and Biotie Therapies Corporation (U.S. Pat. No. 6,624,202). Examples from these series of inhibitors were shown to be effective in a number of in vivo inflammation models, such as mouse ulcerative colitis, mouse LPS-induced septic shock, rat carrageenan foot models, in a mouse model that resembles human multiple sclerosis, in various rodent models of arthritis, and in a transient forebrain ischemia model in estrogen-treated ovariectomized female rats. Some of these hydrazine-based inhibitors were reported to show selective inhibition of SSAO/VAP-1 compared to MAO. However, these compounds are not necessarily desirable therapeutic compounds since the hydrazine functional group is frequently associated with undesirable side effects.

Other families of SSAO/VAP-1 inhibitors have appeared recently in the scientific and patent literature and these are also described in the review cited previously (McDonald I. A. et al.). Some of these compounds, such as the thiazole heterocylic series from Astellas, are reported to be very potent against the rat and human enzyme. One example is shown below. It is reported that these did not inhibit MAO. Another compound was claimed to inhibit damage to vascular permeability in the eyes of streptozotocin-induced diabetic rats.

Haloallylamine compounds that differ from Mofegiline in core structure have been synthesized and were shown to inhibit the amine oxidase activity from copper-dependent amine oxidases from a number of species (see Kim J. et al., Inactivation of bovine plasma amine oxidase by haloallylamines. Bioorg Med Chem 2006, 14, 1444-1453). These compounds have been included in a patent application (Sayre, L. WO 2007/005737).

A number of publications discuss these and other amine oxidase inhibitors, but none of these inhibitors were tested as inhibitors of human SSAO/VAP-1 (see Lee Y et al., 3-Pyrrolines are mechanism-based inactivators of the quinone-dependent amine oxidases but only substrates of the flavin-dependent amine oxidases. J Am Chem Soc 2002, 124, 12135-12143; Zhang Y. et al., Highly potent 3-pyrroline mechanism-based inhibitors of bovine plasma amine oxidase and mass spectrometric confirmation of cofactor derivatization. Bioorg Med Chem 2007, 15, 1868-1877).
2-Halo-substituted-4-amino-2-butenoic acid and ester derivatives are known in the literature (see, for example, Chebib M., et al., Analogs of γ-aminobutyric acid (GABA) and trans-4-aminocrotonic acid (TACA) substituted in the 2 position as GABAC receptor antagonists. Brit J Pharmacol, 1997, 122, 1551-1560; Silverman R. B. and George C., Inactivation of γ-aminobutyric acid aminotransaminase by (Z)-4-amino-2-fluorobut-2-enoic acid. Biochemistry, 1988, 27, 3285-3289). These compounds are described in the patent application WO2007/005737 (Amine oxidase inhibitors) where they are claimed to inhibit copper-dependent amine oxidases and methods of using such inhibitors for therapeutic applications. There was no recognition, however, of the inhibitory effects of these compounds against human SSAO/VAP-1.

3-Adamantyl-substituted 3-chloroallyamine has been reported in the literature, but there is no indication, however, of whether this compound inhibits SSAO/VAP-1 or any other amine oxidase enzyme (Vashkevich E. V. et al., Synthesis of trichloronitrodienamino adamantine derivatives. Russian J Applied Chem, 1999, 35, 1773-1776; Vashkevich E. V. et al., Synthesis of surfactants derived from adamantane. Russian J Applied Chem, 2001, 74, 1892-1989).